The epidemic of obesity is associated with detrimental metabolic complications. Critical to our understanding of these metabolic complications is delineating the mechanisms that regulate the metabolic fate of fatty acids. When fatty acids enter cells they are converted to acyl-CoAs by the actions of acyl-CoA synthetases (ACSL). Emerging data suggests that each of the five ACSL isoforms directs acyl-CoAs to specific metabolic fates. In this grant application we propose to investigate the role of ACSL5 in regulating intestinal and liver triacylglycerol metabolism. ACSL5 is highly expressed in both the intestine and liver, yet our understanding of its functions in these tissues is limited. T address the role of ACSL5 in cell-specific and systemic metabolism we have generated two different lines of ACSL5 knockout mice: one in in which the gene has been constitutively disrupted in all tissues (null) and a second conditional knockout (Acsl5loxP/loxP) that allows us to disrupt ACSL5 expression in specific tissues. In the first specific aim of this application, we propose to generate mice with an intestine-specific ablation of ACSL5 in order to determine: 1) the role of intestinal ACSL5 on enterocyte fatty acid trafficking and, 2) the effects of intestinal ACSL5 on whole body metabolism and signaling, and alterations in gut microbiota 3) the mechanistic basis by which enterocyte ablation of ACSL5 modulates energy metabolism and body fat accumulation. To perform these studies, we will mate our line of conditional ACSL5 knockout mice (Acsl5loxP/loxP) to mice expressing Cre driven by a villin promoter to specifically ablate ACSL5 expression in the intestine (Acsl5int-/-). In a second specific aim, we will determine the role of ACSL5 in regulating hepatic lipid and whole-body energy metabolism and insulin resistance. Obesity and energy dense diets result in increased ACSL5 expression in livers. We hypothesize ACSL5, is upregulated in the liver by SREBP transcription factors, known to induce hepatic steatosis because it catalyzes the formation of acyl-CoAs that are utilized by mtGPAT1 the rate limiting enzyme in lipogenesis. In this aim we will generate mice in which ACSL5 expression is ablated specifically within hepatocytes (Acsl5L-/-). We will feed our line of Acsl5L-/- mice a high fat, high sucrose diet to determine how liver specific ablation of ACSL5 regulates hepatic lipid metabolism, including TAG accumulation and insulin-glucose homeostasis, and determine whether loss of ACSL5 direct fatty acids to other ACSL isoforms and oxidation. We will overexpress SREBP-1c and mtGPAT in our newly developed line of Acsl5L-/- mice to determine the role of hepatic ACSL5 expression in mediating the lipogenic actions of these two proteins. Lastly, we will determine the ability of ACSL5 ablation to alleviate steatosis in ob/ob mice. We expect these studies will result in major advancements into our understanding of fatty acid metabolism and, thereby, provide novel therapeutic avenues for the treatment of obesity, hepatic steatosis and related diseases.